Online Resource 1. Clinical details of King Penguins (Aptenodytes patagonicus) found along the coast and/or maintained in captivity in South Africa, 2001-2017.

Beach-cast king penguin – Record E

10 Aug 2005 –Adult-plumage bird was found at Camps Bay beach, Western Cape (33°57'S, 18°22'E), and sent to the Cape Town rehabilitation facility. Body mass was 9.7 kg.

11 Aug 2005 – The bird was found dead in the morning. Post-mortem examination revealed that a long-line fishing hook had penetrated the stomach wall and liver, causing extensive hemorrhaging. Female. No blood smears were evaluated.

Beach-cast king penguin – Record F

19 Jan 2007 – Adult-plumage bird was found at Thyspunt, Eastern Cape (34°12'S, 24°43'E), and sent to the Cape St. Francis rehabilitation facility. Body mass was 11.4 kg. Preventative treatment for avian malaria (primaquine 0.5mg/kg PO q24h, trimethoprim-sulphamethoxazole 20mg/kg PO q24h) was administered for 7 days, and antimicrobial treatment (enrofloxacin 5 mg/kg PO q24h) was administered for 5 days.

22 Jan 2007 – Bird started to molt.

24 Jan 2007 – A blood smear was obtained and was negative to parasites.

02 Feb 2007 – An abscess on the right shoulder was lanced and cleaned. Antimicrobial treatment (doxycycline 12 mg/kg PO q24h, amoxycillin 20 mg/kg PO q12h) was administered for 14 days.

07 Feb 2007 – A blood smear was obtained and was negative to parasites.

23 Mar 2007 – A blood smear was obtained and was positive to Babesia sp. with low parasitaemia. Bird presented no clinical signs of illness.

13 Apr 2007 – A blood smear was obtained and was positive to Babesia sp. with low parasitaemia. Bird presented no clinical signs of illness.

24 Apr 2007 – A blood smear was obtained and was negative to parasites.

26 Jun 2007 – A blood smear was obtained and was negative to parasites.

03 Aug 2007 – A blood smear was obtained and was negative to parasites.

26 Dec 2007 – A blood smear was obtained and was positive to Babesia sp. with low parasitaemia. Bird presented no clinical signs of illness, but antiprotozoal treatment (primaquine 0.5mg/kg PO q24h, trimethoprim-sulphamethoxazole 20mg/kg PO q24h) was administered for 10 days.

09 Jan 2008 – Having presented no clinical signs of illness and after obtaining negative serological results for Newcastle disease virus and influenza A virus tests, and with the absence of parasites on coproparasitology and blood smears, the bird was transferred to Henry Doorly Zoo (Omaha, Nebraska, USA). No disease or mortality has been reported since then.

Beach-cast king penguin – Record H

04 Oct 2007 – Adult-plumage bird was found at Milnerton beach, Western Cape (33°52'S, 18°29'E), and sent to the Cape Town rehabilitation facility. Body mass was 15.1 kg. The bird was in good body condition but was coughing and had an ocular discharge. Antimicrobial treatment (amoxycillin 200mg/kg PO q8h, enrofloxacin 20mg/kg PO q12h) was administered for 7 days. A blood smear was obtained and was negative to parasites.

05-13 Oct 2007 – Lethargy, hyporexia and regurgitation were noted; these were presumed to be secondary effects of antibiotic treatment. Pox-like lesions became apparent on the eyelids of both eyes during the same period. Topical antimicrobial treatment (chloramphenicol 10 mg/g q8h) was administered and the bird was maintained in a netted enclosure with an insect repellent.

15 Oct 2007 – A blood smear was obtained and was negative to parasites.

18 Oct 2007 – The bird’s behavior and appetite generally improved; pox-like lesions gradually receding.

22 Oct 2007 – A blood smear was obtained and was negative to parasites.

05 Nov 2007 – Pox-like lesions completely cleared. A blood smear was obtained and was negative to parasites.

12 Nov 2007 – A blood smear was obtained and was negative to parasites.

17 Nov 2007 – Loss of appetite noted.

20 Nov 2007 – A blood smear was obtained and was negative to parasites.

29 Nov 2007 – Lethargy and hyporexia worsened. Antifungal (itraconazole 10 mg/kg PO q24h) and antimicrobial treatment (enrofloxacin 20 mg/kg PO q12h) administered for 12 days.

10 Dec 2007 - The bird was found dead. Post-mortem examination revealed moderate generalized congestion, splenomegaly, hepatomegaly with pale, friable liver, kidneys slightly swollen and pale brown in color and the heart had a small amount of inspissated purulent material between the apex and the pericardium. Male. A post-mortem blood smear revealed intense parasitemia by Babesia peircei. Histopathology showed large numbers of Babesia sp. within erythrocytes in all examined tissue samples, in addition to mild to moderate heterophilic and necrotizing inflammation in multiple organs including the spleen, liver, kidneys, adrenal, skeletal muscle, lungs, small intestine, and pericardial sac. There was a severe, diffuse lymphoplasmacytic inflammation of the small intestine with vasculitis. The primary cause of mortality was determined to be a result of the systemic inflammation. It is unclear if the Babesia sp. alone caused the diffuse inflammation and no bacterial colonies were evident in histologic sections. Additionally, there were multifocal moderate areas of granulomatous steatosis with giant cells surrounded by a yellow homogenous material (presumed ceroid) that may be suggestive of an area of trauma.

Beach-cast king penguin – Record I

12 Feb 2009 – Adult-plumage bird was found at Eerste River mouth, Eastern Cape (34°04'S, 24°13'E), and sent to the Cape St. Francis rehabilitation facility. Body mass was 11.6 kg. The bird was in fair body condition but presented slash wounds on the right foot. Antimicrobial (doxycycline 12 mg/kg PO q24h, amoxycillin 20 mg/kg PO q12h) and antifungal treatment (fluconazole 6 mg/kg PO q24h) were administered for 7 and 21 days, respectively.

14 Feb 2009– Bird started to molt.

17 Feb 2009 – A blood smear was obtained and was positive to Babesia sp. with moderate parasitemia (10-15%) and presented no clinical signs of illness.

24 Feb 2009 – Preventative treatment for avian malaria (primaquine 0.5mg/kg PO q24h, trimethoprim-sulphamethoxazole 20mg/kg PO q24h) administered for 7 days.

03 Apr 2009 – A blood smear was obtained and was negative to parasites.

14 Apr 2009 – The bird presented no clinical signs of illness and the foot injury healed satisfactorily, and it was transferred to Two Oceans Aquarium (Cape Town, South Africa).

14 May 2009 – Loss of appetite was noted.

19 May 2009 – A blood smear was obtained and was positive to Babesia sp. with high parasitemia (>15%). Hyporexia worsened.

20 May 2009 – Regurgitation was noted.

21 May 2009 – Marked hyporexia, lethargy and regurgitation. The bird was transferred to the Cape Town rehabilitation facility to receive intensive veterinary care.

22 May 2009 – Antiprotozoal treatment (primaquine 1 mg/kg PO q24h) initiated.

23 May 2009 – The bird was found dead. Post-mortem examination revealed marked generalized congestion, marked lung edema, mild hydropericardium, marked hepatomegaly with pale liver, marked splenomegaly, enlarged gall bladder, kidneys moderately swollen and pale brown in color, marked hyperplasia of the red bone marrow and multifocal petechiae of the abdominal fat. Male. A post-mortem blood smear revealed intense parasitemia by Babesia sp. Histopathology showed large numbers of Babesia sp. within erythrocytes. There was severe congestion and edema with fibrin thrombosis in the lungs. The kidneys showed moderate nephrosis and fibrin thrombosis of glomerular blood vessels with multifocal extra-medullary hematopoesis. There was moderate bile stasis and fibrin thrombosis of portal and centrilobular veins in the liver. Babesiosis was considered the primary cause of death.

Beach-cast king penguin – Record J

22 Feb 2016 – Adult-plumage bird was found in the sand dunes at St. Lucia beach, KwaZulu-Natal (28°23’S, 32°25’E). The bird appeared weak and lethargic, and appeared to be struggling during a very hot summer afternoon (when temperatures in excess of 30°C are not uncommon). The penguin was captured and taken into a facility with air conditioning, and appeared to recover temporarily. However, it died in the evening of the same day during car transportation to Durban. Body mass was 12.45 kg and there was no evidence of pre-molt. The carcass had to be frozen due to logistical constraints. Post-mortem examination revealed good body condition, generalized congestion of the carcass, severe generalized congestion and hemorrhage in the lungs with areas of atelectasis, local extensive mucosal esophageal hemorrhage, mild hepatomegaly and congestion, mild tricuspid valvar endocarditis, and moderate kidney congestion. Male. The stomach contained a moderate quantity of doughy yellow-green soft contents with a small number of squid beaks and stones. Histopathology showed moderate lymphoplasmacytic enteritis, multifocal hyaline degeneration with myofiber swelling, diffuse spleen congestion, and hepatic hemosiderosis. No blood smears were evaluated. Freezing artefacts precluded a conclusive assessment as to whether there were protozoal parasites (e.g. Babesia or Plasmodium). Cause of death could not be confidently established, but heat stress was considered plausible.

Beach-cast king penguin – Record K

27 Apr 2017 – Adult-plumage bird was found at Hout Bay beach, Western Cape (34°03’S, 18°21’E), and sent to the Cape Town rehabilitation facility. Body mass was 9.88 kg. The bird’s plumage was worn-out and discolored, indicating an arrested molt. There was a through-and-through cut (c. 2 x 1 cm) to the oral mucosa and skin directly under the left side of the mandible. This lesion had re-epithelized margins, and was considered likely to have been caused by a fishing hook.

28 Apr 2017 – A blood smear was obtained and was negative to parasites. Packed cell volume was 55% and total plasma protein was 10.4 g/dL, indicating dehydration. Bird had excellent appetite.

04 May 2017 – A blood smear was obtained and was negative to parasites. Packed cell volume was 41% total plasma protein was 8.6 g/dL.

08 May 2017 – Bird started to molt and appetite started to decrease. A blood smear was obtained and was negative to parasites. Packed cell volume was 37% and total plasma protein was 7.2 g/dL.

15 May 2017 – At 7:00 AM, regurgitated fish was found in pen and bird was found showing signs of dyspnea. At 8:20 AM, bird was found dead. Post-mortem examination revealed mid-molt stage, excellent body condition, generalized congestion of the carcass, mild congestion of air sacs, severe congestion of the lungs, moderate hepatomegaly and liver congestion, multifocal pale spots (1-3 mm) randomly distributed throughout the liver parenchyma, moderate splenomegaly, small quantity of food material and mucus in the proximal trachea, and small blood clots (c. 0.2 mL) in the distal trachea. Male. Histopathology showed moderate to severe lymphoplasmacytic enteritis, moderate to severe mixed cholangiohepatitis with random multifocal acute hepatic necrosis, diffuse lung congestion with extensive interstitial and airway hemorrhage, diffuse heart congestion with multifocal interstitial hemorrhages, diffuse adrenal congestion with multifocal interstitial hemorrhages, diffuse kidney congestion with moderate tubular nephrosis, and diffuse brain congestion with vasogenic edema. Bacterial cultures of the left thoracic air sac did not produce bacterial growth, and normal flora (no pathogenic bacteria) was isolated from the duodenal lumen. A post-mortem blood smear revealed a high percentage (>60%) of erythrocytes presented Rickettsia-like cytoplasmic inclusions. Kidney impression smears were negative for protozoal meronts. Cause of death was considered to have been bacterial enteritis with complicating septicaemia (possibly related to the Rickettsia-like infection), in association with arrested molt and agonal aspiration.

Permanently captive king penguin – Case 1

Jan 2009 – Adult-plumage bird was admitted to the Two Oceans Aquarium (Cape Town), having been transferred from the National Zoological Gardens of South Africa (NZG – Pretoria, South Africa). No parasites had been seen on blood smears evaluated while the penguins were at the NZG. Following admission to TOA, blood smears were obtained monthly and were negative to parasites.

30 Jun 2009 – The bird was found dead. Post-mortem examination revealed severe, subacute, proliferative pneumopathy causing acute respiratory failure and post-mortem blood smear did not reveal any blood parasite infection.

Permanently captive king penguin – Case 2

Jan 2009 – Adult-plumage bird was admitted to the Two Oceans Aquarium (Cape Town), having been transferred from the NZG. No parasites had been seen on blood smears evaluated while the penguins were at the NZG. Following admission to TOA, blood smears were obtained monthly and were negative to parasites.

21 Jan 2011 – A blood smear was obtained and was positive to spirochetes that were morphologically consistent with Borrelia sp.

22 Jan 2011 – The bird was found dead. Post-mortem examination revealed generalized congestion and extensive petechiation over the pericardium, myocardium, air sacs, liver capsule and mesenteric fat. The liver and spleen were enlarged and congested. Histopathology showed moderate congestion of organs with necrosis and apoptosis of hepatocytes, mild leucostasis and fibrin thrombosis in the lungs, mild lymphoplasmacytic interstitial nephritis and multifocal villi necrosis in the small intestine. Silver staining (Warthin-Starry) revealed large numbers of spirochetes in the spleen, liver, lung and kidney. The primary cause of mortality was determined to be spirochetosis.

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